Affectivity of Spodoptera littoralis nucleopolyhedrovirus ( Spli NPV ) against first and second instar larvae of the cotton leafworm , Spodoptera littoralis ( Boisd . ) ( Lepidoptera : Noctuidae )

Preliminary bioassays were conducted to determine the virus activity against the 1 st and 2 nd instar larvae of the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). First instar larvae were more susceptible to the virus than the second instar larvae. Mortality response of the larvae was greater at the highest concentration of the virus than at lowest concentrations. The lethal concentration that killed 95% of the tested larvae (LC95%) was 2.32 x 10 6 . The obtained mortality rates of S. littoralis first instar (1 st ) larvae were 0, 15.15, 16.77, 43.57, 82.44 and 96.94%, for the tested concentrations of control (2 x 10 2 , 2 x 10 3 , 2 x 10 4 , 2 x 10 5 and 2 x 10 6 PIB/ml). However, second instar (2 nd ) larvae were less susceptible to the virus than the first instar larvae with the mortality rate of 0, 4, 9.39, 25.33, 43.24, and 71.63%, for the same tested concentrations, respectively. In control treatment, no mortality was recorded. The high concentration levels caused a high rate of mortality. The same trend of mortality is similar to those found by other studies.


INTRODUCTION
The increased awareness of environmental pollution and the demand for safe food production have led to growing interests in use of biological control agents such as baculoviruses in plants' protection.Spodoptera littoralis (Boisd.) is an important key pest on clover and many vegetable crops in Saudi Arabia (Albarrak, 2009).In fact, the cotton leafworm is a major limiting factor affecting crop and vegetable production in many other countries.S. littoralis is one of the most destructive agricultural lepidopterous pests within its subtropical and tropical regimes (Hosny et al., 1986).It can attack numerous economically important crops all year round.Chemical is used for controlling the species of S. littoralis.This control has been used especially on cotton.Up to 1968, methyl-parathion was used for S. littoralis, but then resistance to this compound developed.The chemicals that have been used such as organophosphorus, pyrethroid and other insecticides have appearance of resistance and cross resistance in many cases (Issa et al., 1984).The control of this insect is usually done by excessive use of chemical pesticides which may lead to the development of pesticide resistance.Also, the excessive use of chemicals will lead to negative impacts on the environment and non-target species.*Corresponding author.E-mail: kokodwisutanto@yahoo.com or ksutanto@ksu.edu.sa.Tel: +966537102956.
The microbial control agents in pest management such as viruses are environmental friendly, as well as other microbial control agents such as bacteria, fungi, and nematode; but for their successful use, a specialist's knowledge is needed (Lacey and Kaya, 2000).
Baculoviruses are promising alternatives for reducing dependency on harmful chemical insecticides in plant protection.They are safe, specific, environmental friendly bio-agents, and effective against certain insect pests (Burges et al., 1980).These viruses are specially designed to survive outside their host and can persist in crevices and soil for years (Millar, 1997).They have the greatest potential for use as microbial insect pest control agents.Therefore, Szewczyk et al. (2009) described that these viruses are excellent for their species specificity as biopesticides.
SpliMNPV is a means of controlling cotton leafworm, S. littoralis (Boisd.).The objective of this study is to provide a clearer understanding of the product, which makes instars larvae of S. littoralis more susceptible to the virus (SpliMNPV) under laboratory condition.

Source of test insect
S. littoralis, female moths, were collected by light trap from Alwaseel, Riyadh, Saudi Arabia.Collected moths were transferred to plastic cages with piece of cotton wetted with sugar solution (10%) for egg laying in the laboratory.The obtained eggs from the moths were kept in plastic containers until hatching.

Mass rearing of the cotton leafworm
The colony of the cotton leafworm was established and maintained at the rearing laboratory at the Economic Entomology Research Unit (EERU), Plant Protection Department, King Saudi University.The laid egg masses were transferred into plastic cups, and covered with tissue paper until egg hatching.Neonate larvae were reared in group in plastic cups with a thin layer of semi-artificial diet described by Shorey and Hale (1965).The semi-artificial diet described by Shorey and Hale (1965) contained a mixture of 250 g white bean, 20 g agar, 15 g ascorbic acid, 1.2 g sorbic acid, 80 g yeast, 5 g natrium benzoate and 1200 ml distilled water.All ingredients were cooked (except of ascorbic acid) and mixed, then poured into plastic cups (D: 6.2 cm); ascorbic acid was added when the mixture cooled down at 60°C and then poured in plastic cups and stored at 5°C.Third instar larvae were reared individually to pupation using the same diet in plastic cups, to avoid cannibalism.Pupae were transferred to a plastic box (L: 18 cm, W: 12 cm, and H: 6.5 cm) until emergence.Five pairs of moths were transferred to each jar and supplied with 10% sugar solution for mating and producing fertilized eggs.The colony was maintained under controlled conditions of 25 o C and 60-70% RH.

Virus inocula
The virus source is the commercial product Littovir®.It was obtained from Biocontrol, Switzerland, Distributer, AlRashed est.for Trading and Agriculture, Riyadh, Saudi Arabia.Polyhedral Inclusion Body (PIB) of SpliMNPV was 2x10 12 PIB/L (Ralf- Udo et al., 2008).Dilutions of virus suspensions were prepared, as required by dilution in a glass tubes (Lab.Glass, India).

Bioassay for determination of lethal concentrations (LC) values of SpliMNPV
In order to determine the lethal concentration of SpliMNPV at 95-99%, diet surface treatment bioassays were conducted, in which six viral concentrations of 2 x 10 2 , 2 x 10 3 , 2 x 10 4 , 2 x 10 5 and 2 x 10 6 PIB/ml were tested against first and second instar larvae of S. littoralis.In this bioassay, the artificial diet was spread in bioassay plates (5 x 10 x 1 cm) (LICEFA, Bad Salzuflen (DE), Germany), divided into 50 cells using special cover plate.One milliter of each virus treatment was spread on the surface of artificial diet in each bioassay plate.After dryness of the viral film, fifty larvae of each instar were transferred into plate cells individually.The bioassay plate was covered by two layers of tissue paper and glass cover fixed with rubber band to prevent larvae from escaping.In untreated control treatment was used distilled water only.The experiment was incubated at 25°C and 70% R.H. Larval mortality was recorded daily up to 15 days, and LC value was calculated according to probit analysis (Finney, 1971).
Larval mortality was observed daily according to the following initial signs: gradual changes in colour (cuticula showing a pale, whitening colour) and milkiness (Tanada and Kaya, 1993).

Mortality response of larvae S. littoralis to SpliMNPV concentrations
In order to determine the lethal concentration (95-99%) of SpliMNPV that kill the tested first and second instar of S. littoralis larvae, five successive concentrations were tested.Data presented in Table 1 shows that first instar larvae were more susceptible to the SpliMNPV.
The mortality rates of S. littoralis first instar (1 st ) larvae (Table 1) were 0, 15.15, 16.77, 43.57, 82.44, and 96.94% respectively, at concentration levels of 2 x 10 2 , 2 x 10 3 , 2 x 10 4 , 2 x 10 5 and 2 x 10 6 PIB/ml, respectively.However, second instar (2 nd ) larvae showed to be less susceptible to the virus than the first instar larvae with the mortality rate of 0, 4, 9.39, 25.33, 43.24, and 71.63%, for the same viral concentration, respectively.No mortality was recorded in case of the untreated control.The obtained result of high rate of mortality depends on the concentration levels, is similar to that found by Ignoffo (1966).
Related to this result, Wilson et al (2000) also mentioned that second instar more susceptible to AcMNPV than four instar larvae of cabbage looper, Trichoplusiani.Also, Cherry et al., (2000) reported that the susceptibility of Helicoverpha armigera depends on the larvae age when HearNPV applied.Also mentioned that earlier instar has speed of kill by AsNPV than older instar larvae of Agrotis segetum (Bourner et al., 1992), and CfMNPV on spruce budworm larvae (Duan and Otvos, 2001).These bioassays determined SpliMNPV concentration, which caused percentage mortality of 90   and 95% based on Shapiro (1992) study.From this study, the calculated lethal concentration at 95% (LC 95 ) was 2.32x10 6 PIB/ml, which was used for the further UV exposure tests in the presence or absence of the additives.

Instars of larvae
The obtained viral symptoms shown in Figure 1, is typical to that described by Tanada and Kaya (1993).The sign of infected larvae by virus was pale color, milky and at the end, the body of larva was melted.The death of S. littoralis larvae due to SpliMNPV appears in approximately 8-8.5 days after treatment for third instars and in approximately 3-3.5 days for neonates (Toprak et al., 2005).
The virus infection occurred when larvae ingested the Polyhedral Inclusion Body (PIB) and it reached the insect's midgut (Moore and Manousis, 1986).According to Petrik et al., (2003), releasing of SpliMNPV virions (OBv) in the midgut were infectious to the larvae, they entered and spread in the midgut in order to cause the infection.
The tested larvae were highly sensitive and susceptible.
The highest concentration of the virus caused great mortality to the larvae rather than lower concentrations within a short time of 4 days after application.The young larvae can be killed by virus within 2-4 days (Ignoffo, 1966) cit.(Toprak et al., 2005).The potential of S. littoralis MNPV as a biological control has been described; also this virus has potential to control beet armyworm S. exigua, and fall armyworm S. fregiperda (Simón et al., 2004).Another virus such as SeMNPV is only able to cause infection in beet armyworm (S. exiqua) (Yanase et al., 1998b?).In case of this experiment, the susceptibility of cotton leafworm, S. littoralis to SpliMNPV occurred especially in young instar larvae.This is similar to the result of AfMNPV and AcMNPV when this virus was applied to the neonate larvae of codling moth (Lacey et al., 2002).Virus infection in insect larvae occurs within 5-15 days.Liquefied symptom is the character of virus infection, because virus spread in tissues and organs of the larvae (El Salamouny, 2007).
In the control group, distillated water was used; it was shown that there was no mortality.It was concluded that control (distilled water) and treatments (virus concentrations) were significantly different and LC95% of virus concentration was 2.32 x 10 6 PIBs/ml (Figure 1).

Conclusion and recommendation
First instar larvae of S. littoralis were highly susceptible to the SpliMNPV than the second instar larvae; the highest concentrations caused high rate of mortality and 2 x 10 6 is the most effective concentration of larvae mortality.
SpliMNPV is the greatest microbial insect pest control agent but the activity of this virus is still limited due to the degradation of its activity under field conditions (UV radiation).Therefore, there is need to have some virus protection, such as natural UV protectant (based on some plant derived materials), to increase the persistence of this virus in adverse harsh sunny conditions in the Kingdom of Saudi Arabia, for it to be effective.
of SpliMNPV of diet surface.3 replicates; 50 larvae per treatment per replicates; 50 larvae untreated control per replicate.b Instar of S. littoralis are 1 st and 2 nd instar larvae.c Means within columns with the same letter are not significantly different (P>0.05) by LSD.

Figure 1 .
Figure 1.The susceptibility of first and second instars larvae to SpliMNPV measured by lethal concentration (LC) value.

Table 1 .
Mortality responds of S.littoralis larvae to different concentration of SpliMNPV (Means ± SE) a,b,c .